Selective Head Cooling and NOX Inhibition Protect the Blood-Brain Barrier in Neonatal Epilepsy

Epileptic seizures in the neonatal brain induce oxidative stress and disrupt the blood-brain barrier (BBB), leading to long-term cerebrovascular and neurodevelopmental deficits. This study examined the protective effects of selective head cooling and NADPH oxidase (NOX) inhibition on BBB integrity following seizures. Neonatal seizures were induced in newborn pigs with bicuculline under normothermic or selective head cooling conditions. BBB disruption was assessed by Evans Blue extravasation and quantification of circulating brain-derived endothelial cells (CD45^-/CD146⁺/GluT1⁺). Seizures under normothermia caused marked BBB leakage, cerebrovascular apoptosis, and elevated endothelial biomarkers, whereas selective head cooling (cortical temperature ~25 °C, body ~35 °C) significantly reduced these effects. Pharmacological inhibition of NOX with setanaxib (5 mg/kg) or sulforaphane (0.4 mg/kg) also prevented BBB disruption during normothermia. In vitro, primary porcine and human brain endothelial cells exposed to glutamate or TNF-α showed increased NOX activity, ROS production, apoptosis, and barrier leakage, all attenuated by NOX inhibitors or moderate hypothermia (<30 °C). These findings identify endothelial NOX as a key mediator of seizure-induced BBB injury and demonstrate that both NOX inhibition and selective head cooling effectively preserve cerebrovascular integrity. Combined hypothermic and antioxidant therapy may offer a promising strategy to prevent cerebrovascular injury and BBB damage in neonatal epilepsy.